DESCRIPTION: (Scanned from the applicant's description) Insulin stimulates glucose transport in fat and muscle cells by inducing the redistribution of a tissue specific glucose transporter, GLUT4, from an intracellular membrane pool to the plasma membrane. The mechanisms underlying this effect of insulin have been difficult to elucidate. Insight into the nature of these mechanisms would be facilitated by direct observation, in live cells, in real time, of the dynamic process of GLUT4 traffic. To this end, we have begun to analyze GLUT4 trafficking with a unique technology, originally developed for the imaging of calcium signals in isolated muscle cells, called Ultra-Fast Digital Imaging Microscopy. The key advantage of this technology is that it enables the visualization of a fluorophore within the whole 3 dimensional volume of the cell at high frequency (intervals as short as 3 msec) using extremely low light exposure times. In this proposal we present four-dimensional images (3-D images over time) of GFP-GLUT4 under basal conditions and in response to insulin, acquired using this technology. The images suggest several previously unidentified mechanisms of vesicular transport that may be important for insulin action on GLUT4 translocation. For example, we find that the accumulation of GLUT4-eGFP at the plasma membrane is preceded by movement of vesicles from a juxtanuclear region to the plasma membrane with direction and speed consistent with a microtubule-mediated transport mechanism. In addition, vesicles are targeted to specific locations on the plasma membrane, by a mechanism that involves actin filament integrity. Based on these results we propose to test the following hypotheses: SPECIFIC AIM 1: To test the hypothesis that insulin-stimulated movement of GLUT4 to the plasma membrane depends on microtubule-based motors SPECIFIC AIM 2: To test the hypothesis that GLUT4 traffic also involves interaction with the actin cytoskeleton through specific myosin motors. SPECIFIC AIM 3: To test the hypothesis that the fusion sites of GLUT4 with the plasma membrane are defined by the mammalian homologues of the yeast "exocyst." SPECIFIC AIM 4: To test the hypothesis that insulin stimulates budding of GLUT4 vesicles from the juxtanuclear region by imaging at higher spatial and temporal resolution.